Species specificity of the NS1 protein of influenza B virus: NS1 binds only human and non-human primate ubiquitin-like ISG15 proteins

Abstract

Influenza B viruses, which cause a highly contagious respiratory disease every year, are restricted to humans, but the basis for this restriction had not been determined. Here we provide one explanation for this restriction: the species specificity exhibited by the NS1 protein of influenza B virus (NS1B protein). This viral protein combats a major host antiviral response by binding the interferon-alpha/beta-induced, ubiquitin-like ISG15 protein and inhibiting its conjugation to an array of proteins. We demonstrate that the NS1B protein exhibits species-specific binding; it binds human and non-human primate ISG15 but not mouse or canine ISG15. In both transfection assays and virus-infected cells, the NS1B protein binds and relocalizes only human and non-human primate ISG15 from the cytoplasm to nuclear speckles. Human and non-human primate ISG15 proteins consist of two ubiquitin-like domains separated by a short hinge linker of five amino acids. Remarkably, this short hinge plays a large role in the species-specific binding by the NS1B protein. The hinge of human and non-human primate ISG15, which has a sequence that differs from that of other mammalian ISG15 proteins, including mouse and canine ISG15, is absolutely required for binding the NS1B protein. Consequently, the ISG15 proteins of humans and non-human primates are the only mammalian ISG15 proteins that would bind NS1B.

FIGURE 1.

The NS1B protein exhibits species specificity in binding ISG15. The indicated cells were co-transfected with plasmids expressing the NS1B protein and GST alone (lanes 1, 6, and 11), GST-human ISG15 (lanes 2, 7, and 12), GST-mouse ISG15 (lanes 3, 8, and 13), GST-canine ISG15 (lanes 4, 9, and 14), or GST-monkey ISG15 (lanes 5, 10, and 15). Cell extracts were subjected to glutathione-Sepharose selection, and the eluents were analyzed by immunoblots (WB) using anti-NS1B (top panel) or anti-GST antibody (middle panel). Aliquots of the cell extracts containing equal protein amounts were analyzed by an immunoblot using anti-NS1B antibody (bottom panel). MDCK, Madin-Darby canine kidney cells.

FIGURE 2.

NS1B alters the intracellular localization of only human and monkey ISG15. A, HeLa cells were co-transfected with plasmids encoding GFP-NS1B and 3xFLAG-human ISG15 (middle column) or 3xFLAG-mouse ISG15 (right column). As a control, cells were co-transfected with plasmids encoding GFP and 3xFLAG-human ISG15 (left column). Confocal microscopy was carried out as described under “Experimental Procedures.” The Merge panel shows an overlay of the GFP and rhodamine signals, and the inset shows a magnified image of the boxed cell in the Merge panel. IF, immunofluorescence. B, HeLa cells were transfected with a plasmid expressing the indicated 3xFLAG-ISG15 protein, and 24 h later, the cells were either mock-infected (left column) or infected with influenza B virus (middle and right columns). The white arrows indicate the human and monkey ISG15 molecules that have relocalized to nuclear speckles in infected cells, and the white lollipops indicate the infected cells expressing mouse and canine ISG15.

FIGURE 3.

Critical role of the hinge of human ISG15 in binding the NS1B protein. A, top, schematic representation of ISG15 with the N- and C-terminal domains linked by the hinge (H). HEK 293T cells were co-transfected with plasmids encoding NS1B and the indicated ISG15 proteins fused to GST: human ISG15 (Hum) (lanes 1 and 4), canine ISG15 (Can) (lane 2), canine ISG15 with the human hinge (Can/Hum-H) (lane 3), mouse ISG15 (Mou) (lane 5), and mouse ISG15 with the human hinge (Mou/Hum-H) (lane 6). Cell extracts were analyzed as described in the legend for Fig. 1. WB, immunoblot. B, top, alignment of human and mouse hinge (H) sequences, with the differences between the sequences denoted by asterisks. HEK 293T cells were co-transfected with plasmids encoding NS1B and GST fused to the indicated human ISG15 molecules: wild-type (wt) (lane 1), mouse hinge replacing the human hinge (Mou-H) (lane 2), D76Q mutant (lane 3), K77N mutant (lane 4), and D79S mutant (lane 5). C, alignment of the hinge sequences in the ISG15 molecules of the indicated mammalian species. The human and non-human primate hinge sequences are boxed.